The diagnosis of tuberculosis (TB) is the major weak link in global TB control. Drug resistant TB compounds diagnosis and treatment challenges. The most widely used diagnostic test, sputum smear microscopy, is cumbersome, insensitive, involves multiple patient visits and cannot detect multi drug or extensively drug resistant (MDR/XDR-TB). Sputum culture is costly and depending on the method used takes 4-8 weeks to obtain a result and up to 16 weeks to detect MDR/XDR-TB. Consequently, there is an urgent need for a simple, rapid diagnostic test to identify the presence of Mycobacterium tuberculosis (MTB) and perform MDR/XDR genotyping that could be performed at the public health clinic while the patient waits. The long-term objective of this project is to develop a MTB identification and MDR/XDR-TB genotyping test that can be used at the point-of-care (POC). Investigen's "smartDNATM technology" enables DNA detection to occur rapidly at room temperature in homogenous solution with colorimetric readout. The smartDNA MTB identification assay consists of: (1) conventional sputum treatment, (2) sample disruption, (3) MTB target capture by a peptide nucleic acid (PNA)-microparticle, and (4) detection via a `light-activated'color change of a second (detection) PNA-DNA target bound dye. Investigen plans to extend the use of smartDNA-based detection to MDR/XDR- TB genotyping. The proposed protocol for MTB identification and MDR/XDR-TB genotyping involves (1) conventional sputum treatment, (2) sample disruption, (3) PNA-microparticle enrichment capture of MTB conserved sequences in each of the target areas of interest, (4) sequence specific binding of the enrichment captured targets on elements of an allele-specific PNA array and (5) detection of the resulting PNA-DNA hybrids using smartDNA. The successful development of the POC smartDNA MDR/XDR-TB genotyping technology requires that (1) probe sets for the capture, detection and allele discrimination of mutations involved in MDR/XDR-TB be designed, and (2) a practical way is found to apply the smartDNA dye color change reaction to PNA-DNA hybrids bound to a planar surface. There are three specific aims. (1) Demonstrate utility of the conceived system to differentiate single base changes in MTB genomic DNA. (2) Design probe sets for the detection of mutations. (3) Develop smartDNA readout on a PNA array. A smartDNA-based MDR/XDR- TB genotyping assay would greatly improve the state of MTB diagnosis and treatment, and accordingly, is relevant to the NIH mission of reducing the burden of illness through supporting research for the improved diagnosis, prevention and cure of human diseases. PUBLIC HEALTH RELEVANCE While the United States has been largely successful in detecting, treating and controlling TB, in many countries TB, MDR-TB and XDR-TB are a serious and growing public health issue;international travel and immigration have increased transmission. The research to be completed on this project is anticipated to lead to the development of a TB, MDR-TB and XDR-TB identification system that will provide results while the patient waits and can be used in resource constrained settings throughout the world, in clinics and emergency rooms in the United States and potentially even at US borders greatly aiding public health services in the detection, treatment and control of TB, MDR-TB and XDR-TB.